Article pubs.acs.org/est
Emerging Contaminant or an Old Toxin in Disguise? Silver Nanoparticle Impacts on Ecosystems Benjamin P. Colman,*,†,▽ Benjamin Espinasse,‡,▽ Curtis J. Richardson,§,∥,▽ Cole W. Matson,∥,⊥,▽ Gregory V. Lowry,#,▽ Dana E. Hunt,∥ Mark R. Wiesner,‡,▽ and Emily S. Bernhardt†,▽ †
Department of Biology, Duke University, Durham, North Carolina 27708, United States Civil and Environmental Engineering Department, Duke University, Durham, North Carolina 27708, United States § Duke University Wetland Center, Nicholas School of the Environment, Duke University, Durham, North Carolina 27708, United States ∥ Nicholas School of the Environment, Duke University, Durham, North Carolina 27708, United States ⊥ Department of Environmental Science and the Center for Reservoir and Aquatic Systems Research (CRASR), Baylor University, Waco, Texas 76798, United States # Civil & Environmental Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United States ▽ Center for the Environmental Implications of Nanotechnology, Duke University, Durham, North Carolina 27708, United States ‡
S Supporting Information *
ABSTRACT: The use of antimicrobial silver nanoparticles (AgNPs) in consumer-products is rising. Much of these AgNPs are expected to enter the wastewater stream, with up to 10% of that eventually released as effluent into aquatic ecosystems with unknown ecological consequences. We examined AgNP impacts on aquatic ecosystems by comparing the effects of two AgNP sizes (12 and 49 nm) to ionic silver (Ag+; added as AgNO3), a historically problematic contaminant with known impacts. Using 19 wetland mesocosms, we added Ag to the 360 L aquatic compartment to reach 2.5 mg Ag L−1. Silver treatments and two coating controls were done in triplicate, and compared to four replicate controls. All three silver treatments were toxic to aquatic plants, leading to a significant release of dissolved organic carbon and chloride following exposure. Simultaneously, dissolved methane concentrations increased forty-fold relative to controls in all three Ag treatments. Despite dramatic toxicity differences observed in lab studies for these three forms of Ag, our results show surprising convergence in the direction, magnitude, and duration of ecosystem-scale impacts for all Ag treatments. Our results suggest that all forms of Ag changed solute chemistry driving transformations of Ag which then altered Ag impacts.
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exposed organisms? A number of risk assessments3,4 now suggest that AgNPs are entering wastewaters and are ultimately entering the environment as either wastewater treatment biosolids2 or effluent.5,6 At present, there are no empirical data on silver nanoparticle concentrations and no assessments of their impacts in natural environments. The rapidly expanding toxicological literature on nanomaterial impacts suggests that AgNP pollution has the potential to harm many organisms and that AgNP loading could lead to significant changes in important ecosystem processes, such as primary productivity,7,8 decomposition,9 and nitrogen cycling.10,11 Toxicological research on the environmental impacts
INTRODUCTION
The small size of manufactured nanomaterials (1−100 nm) gives them unique physical and chemical properties, which have led to their use in an ever increasing number of consumer products and industrial processes.1 With this rapid increase in the production and use of nanomaterials, it is inevitable that engineered nanomaterials are entering the environment.2 However, our ability to create and deploy new nanomaterials far outstrips our understanding of what they may do outside of their intended use, leading to concerns that these novel contaminants will have unpredictable and potentially dire consequences when they enter natural environments. Silver nanoparticles (AgNPs) are of particular concern because they are produced specifically for their antimicrobial and biocidal properties. The increasing use of AgNPs leads to two obvious questions: At what concentrations are AgNPs entering the environment? What are their impacts on the © 2014 American Chemical Society
Received: Revised: Accepted: Published: 5229
December 6, 2013 March 18, 2014 April 2, 2014 April 2, 2014 dx.doi.org/10.1021/es405454v | Environ. Sci. Technol. 2014, 48, 5229−5236
Environmental Science & Technology
Article
soils used in previous terrestrial and wetland mesocosm experiments.15,20 Soils were blended and provided by Soils and Sand (Durham, U.S.A.), and were a blend of three soils (“Sandhills”, from Lemon Springs, NC, U.S.A.; “Clay” and “Topsoil”, from Durham, NC, U.S.A.). The final soil had a texture of 63.9% sand, 28.3% silt, and 13.0% clay, with 5.1% loss on ignition. The mesocosms consist of the following: a permanently flooded portion (aquatic zone), a periodically flooded portion (transition zone), and a rarely flooded portion (upland zone). Mesocosms were planted in March of 2010. In the aquatic compartment, six stems of Egeria densa were planted in a two by three grid, Potamogeton diversifolius was unintentionally introduced with the E. densa, and 2 g of the duckweed Landoltia punctata was floated on the water’s surface (Carolina Biological, Burlington, U.S.A.). The transition and upland zones were planted as described in the Supporting Information, SI. Algae and zooplankton were added in 250 mL of unfiltered water per mesocosm from a local wetland. Other organisms were added or were allowed to colonize the mesocosms, as described in the SI. To normalize the water chemistry and algal composition in the mesocosms, a submersible pump was used to circulate water between the 19 mesocosms. Water was circulated using a combination of pumping and siphoning twice prior to dosing in order to ensure reasonable homogenization of the water, as verified by convergence of water chemistry. Experimental Treatments. There were six different treatments overall: Control, received DI water; PVP-coating control, received PVP equivalent to that added with the PVPAgNPs; GA-coating control received gum arabic equivalent to that added with the GA-AgNPs; AgNO3, received sufficient AgNO3 to bring watercolumn concentration to 2.5 mg Ag L−1; GA-AgNPs, received sufficient 12 ± 9.2 nm (mean ± standard deviation) gum arabic coated AgNPs to bring the watercolumn Ag concentrations to 2.5 mg L−1; PVP-AgNPs received sufficient 49.3 ± 22.5 nm PVP-coated AgNPs to reach 2.5 mg Ag L−1. Silver-treated mesocosms all had 900 mg Ag added to bring the watercolumn to 2.5 mg Ag L−1. Since the AgNO3 treatment received a sizable NO3− input, we added an equivalent amount of NO3− to all other treatments in the form of KNO3. The characterization of the AgNP stocks used in this experiment have been previously described.21,22 Treatments were applied mid-day on August 17, 2010, (Day 0) and were applied by pouring treatment water into a funnel with a 10 mL pipet tip on the end of the funnel to slow the flow into the watercolumn. The tip was submerged, but kept within 1 cm of the surface and moved in a grid to add material as uniformly as possible to the watercolumn. The concentration and rate of addition of AgNPs in this experiment are admittedly high, more consistent with existing toxicological literature than they were with predicted environmental exposure scenarios. Additionally, rather than Ag(0)NPs, what has been observed in waste streams has been Ag2SNPs,5,23,24 which have been shown to have diminished toxicity in lab studies25−27 when compared to Ag(0)NPs. While the 2.5 mg Ag L−1 concentration was easily at the low end of the range used in many toxicological studies and is quite low in comparison to concentrations used in many other nanoparticle impact studies conducted to date, it is more similar to untreated photoprocessing effluents than it is to expected concentrations in wastewater effluent, let alone wetlands and streams.5,6,28 In this experiment, these high concentrations were a compromise
of AgNPs, however, has primarily focused on high dose AgNP exposures to single-species cultures grown in well-characterized laboratory media. Although lab studies are essential for elucidating the mechanisms of toxicity, it is quite difficult to extrapolate their findings to natural ecosystems. In natural ecosystems, exposure concentrations are likely to be low, environmental media are both chemically complex and heterogeneous, and the myriad direct and indirect interactions that exist within communities of organisms in natural environments are likely to alter the magnitude and potentially the direction of nanomaterial impacts.12 Up to 10% of the AgNPs entering wastewater treatment plants are predicted to be released in wastewater effluent into aquatic ecosystems,2,5,6,13 thus we chose to explore the impacts of nanoparticle addition in wetlands. To the water column of triplicate 4.5 m2 wetland mesocosms, we added either 12 nm gum arabic coated or 49 nm polyvinylpyrrolidone coated AgNPs (hereafter, GA-AgNPs and PVP-AgNPs, respectively). We compared the fate and impact of these AgNPs to the addition of ionic silver (Ag+) added as AgNO3. Each Ag treatment consisted of a single dose sufficient to raise initial water column Ag concentrations to 2.5 mg Ag L−1. Silvertreated mesocosms were compared to four replicate control mesocosms, and triplicate GA-coating and PVP-coating controls, which received the same amount of the coatings added in the AgNP treatments, only without AgNPs. With one minor exception described in the results, coating controls were identical to control mesocosms. Thus, the focus of the work presented here is on comparisons between controls, GAAgNPs, PVP-AgNPs, and AgNO3 treatments. Dissolved silver is a well-studied contaminant with a long history of environmental impact studies,14 and thus a primary goal of our study was to determine the extent to which the ecological impacts of AgNP exposure differ from the better studied effects of Ag+ exposure. Direct comparisons within individual studies have led to mixed conclusions: some studies found higher toxicity and/or biouptake for AgNPs than AgNO3;7,8,15,16 other studies report the opposite conclusion.17−19 Much of this discrepancy may be explained by differences in AgNP size or coatings; for that reason, we chose to compare the effects of two very different AgNPs with different published toxicity thresholds: the larger PVP-AgNPs are in general less toxic than the smaller GA-AgNPs, which in turn tend to be less toxic than AgNO3.18,19 Thus, we expected to find that dissolved silver would reduce microbial and plant biomass and growth to a greater degree than GA-AgNPs, while both GA-AgNPs and AgNO3 treatments would have stronger impacts than the addition of larger PVP-AgNPs. Furthermore, we expected patterns of bioaccumulation into plant tissues to follow similar trends.
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MATERIALS AND METHODS Setup. The overall design and construction of our slantboard wetland mesocosms were as previously detailed.20 Briefly, treated lumber was used to create 3.66 × 1.22 × 0.81 m boxes. The boxes were partially filled with screening material, and graded to give a flat bottom that was 0.8 m long, connected to a slope upward at an angle of 13° and a run of 2.8 m. Over the screening, an EPDM liner was placed, and over the liner soil was filled to a constant thickness of 22 cm. Mesocosms were built between August of 2009 and March of 2010. The soil used in this experiment was a blend of three topsoils chosen to give a similar texture and organic matter content to 5230
dx.doi.org/10.1021/es405454v | Environ. Sci. Technol. 2014, 48, 5229−5236
Environmental Science & Technology
Article
between expected analytical tractabilitymeasuring the fate, transformation, and impacts of AgNPs in a complex environment over the full course of the 15 month experimentand environmental realism. While actual loading rates and watercolumn concentrations are likely to be much lower, it should also be mentioned that those exposures are likely to be in the form of chronic addition of silver, and so the cumulative load over time may be similar. Additionally, by studying Ag(0)NPs in comparison to Ag+, we are able to better bound the range of what is possible in this worst case scenario, and describe the similarities and differences of AgNP impacts and Ag+ in this complex wetland ecosystem environment. Sampling. For the first 30 days of this experiment, we focused our analyses on the biogeochemistry of the aquatic zone and the treatment effects on plants, phytoplankton, and microbes; these were the organisms that were most abundant, most directly influencing biogeochemistry, and directly affected by the addition of silver. Full details on sampling protocols and sample handling and treatment are available as SI. Briefly, water was collected from 10 cm depth for dissolved solutes, dissolved organic carbon (DOC), and silver concentration. Water for dissolved gases was sampled at the surface using established protocols29 with modifications.30 Vegetation in the aquatic compartment was photographed on Days 2, 7, and 16 from approximately 1 m above the aquatic end of the mesocosms. Analyses. While a subset of water chemistry parameters are highlighted in this paper, water chemistry parameters measured included: the anions Cl−, SO42−, Br−, and NO3−, measured on a Dionex ICS 2000 Ion Chromatograph; NH4+ and o-PO4 measured on a Lachat QuickChem 8500; nonpurgeable dissolved organic carbon (our metric of dissolved organic carbon, DOC) and total dissolved nitrogen (TDN) measured on a Shimadzu TOC-VCPH Analyzer with a TNM-1 module. Dissolved gas samples from headspace equillibrations (see SI) were injected into a Shimadzu 17A Gas Chromatograph using a Tekmar 7050 Headspace Autosampler, as has been previously described. 30 Water column silver was measured on a PerkinElmer 3100 flame-AAS up to Day 8, after which point samples were measured on a PerkinElmer 5100 GF-AAS. Ultrafiltered samples were all measured by GF-AAS. Phytoplankton were enumerated using a BD FACSCalibur Flow Cytometer as previously described.31 Non-photosynthetic prokaryotes were quantified by flow cytometry by first staining the samples with Sybr Green-I as has been previously described.32 Silver speciation was estimated using Visual MINTEQ V3.0 (KTH Royal Institute of Technology, Stockholm, Sweden), which was parametrized using measured solute concentrations and pH. Statistics. In examining solute and gas concentrations over time, we had three questions. First, were there any differences between the true control treatment and the coating controls over time? Second, were there differences between the control(s) and silver treatments over time? Third, were there differences among the silver treatments over time? To answer all of these questions, we conducted two-way ANOVA with time and treatment as factors, and using Holm-Sidak posthoc tests for multiple comparisons within an individual factor.
Figure 1. Silver concentrations declined over time in the water column in (A) total silver (inset is total silver vs 0.7 μm filtered silver); (B) 3 kDa ultrafiltered silver. Samples for 3 kDa ultrafiltered water were not presented past day 6, as values were below our detection limit. The AgNO3 data are represented by black circles, GA-AgNPs by red squares, and PVP-AgNPs by blue triangles. Values are mean ± SEM for n = 3.
GA-AgNPs, PVP-AgNPs, and AgNO3, respectively (Figure 1A, SI Table S1; mean ± standard error of the mean). Total silver concentrations declined most rapidly in the AgNO3 treatment, where Ag concentrations were significantly lower in the first 4 days following dosing (p < 0.05, days 1−4). Despite their size and coating differences, there were no significant differences in water column silver concentration between the two AgNP treatments on any individual date. By day 6, silver concentrations in all Ag treatments had converged to 0.44 ± 0.06 mg Ag L−1. Total silver concentrations were nearly identical to samples filtered through 0.7 μm filters for all three silver forms across all days, with a regression between filtered and unfiltered silver yielding an r2 = 0.993. Between days 8 and 28, Ag concentrations in all Ag treatments remained fairly stable and were similar across treatments (averaging 0.13 ± 0.02 mg Ag L−1). Silver concentrations in the
